Molecular Mechanisms of Atrazine in Altering the Endocrine Gene Network Atrazine (ATR) is widely used as a broadleaf herbicide in the United States and world. While toxic levels of ATR are linked to reproductive abnormalities in wild life, effects in humans remain controversial. Moreover, the molecular and cellular targets of ATR are still unknown. We and others have found that ATR stimulates the nuclear receptor steroidogenic factor 1 (SF-1, NR5A1). In the last decade our lab has worked extensively to define the molecular mechanisms regulating members of subfamily V (NR5A) receptors that includes SF-1, LRH-1, Ftz-f1, ff1a-d. As such, we have begun to ask how ATR functions in mammalian endocrine cell lines and in zebrafish to affect NR5A receptor activity. Our preliminary data show that environmentally relevant ATR concentrations increased the zebrafish (z)Cyp19A1 expression encoding aromatase, and increased the ratio of female to male fish. ZCyp19A1 is a known target of the nuclear receptor SF-1 (NR5A1). By contrast, the second zebrafish gene encoding aromatase zCyp19A2 was unchanged after ATR treatment. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHss, INH1, 1GSU, and 11ss-HSD2. Further cellular studies suggest that ATR increases the activity of mammalian and zebrafish NR5A receptor orthologs by receptor phosphorylation, and amplification of cAMP and investigator3K signaling. Importantly, our data show that ATR does not directly affect SF-1 DNA-binding, nor does ATR appear to directly bind SF-1. Instead, we hypothesize that this environmental herbicide potentially influences endocrine gene networks by indirectly activating NR5A receptors as well as other cAMP-mediated transcriptional events. The precise molecular targets of ATR that result in cell-specific stimulation of NR5A receptors remain to be defined. In this narrowly focused R21 application, two aims are planned to identify the molecular targets of ATR. In our first aim, a systematic genome siRNA screen will be carried out on ATR-responsive reporter cell lines. In our second aim, we will test different categories of candidate targets of ATR. Identifying the underlying mechanism(s) of ATR stimulation of NR5A receptors will begin to address the potentially adverse endocrine disrupting effects of this pervasive and persistent environmental chemical. PUBLIC HEALTH RELEVANCE: Endocrine disrupting chemicals (EDCs) such as the herbicide atrazine (ATR) are widely used and are proposed to adversely affect the endocrine system. Although the consequences of EDCs on the reproductive health and viability of some wild life are established, the precise effects on human health remain controversial. In the United States, use of ATR is heavy in many agriculturally settings, especially in the Midwest where corn is grown, however this herbicide is banned in most EU countries. In this application we seek to define the precise molecular targets of ATR using mammalian culture cells. Our proposed experimental plan to systematically define the molecular targets of ATR is an important step in addressing the potential impact of ATR on wild life and human health and the controversy surrounding its continued use in the US. These studies will also shed new insight into the activation of NR5A receptors as important regulators in reproductive development and physiology.